ISOTOPE EFFECTS AND THE IDENTIFICATION OF CATALYTIC RESIDUES IN THE REACTION CATALYZED BY GLUTAMATE RACEMASE

Primary kinetic isotope effects on V(max) were observed in both reaction directions upon racemizing samples of [2-H-2] glutamate with the cofactor-independent glutamate racemase from Lactobacillus. This supports a deprotonation/protonation mechanism for racemization in which the breaking of the carbon-hydrogen bond at C-2 is partially rate-determining. Substantial ''overshoots'' were observed when the time course of racemization of either enantiomer of glutamate was monitored using circular dichroism spectroscopy. This is consistent with a ''two-base'' mechanism accompanied by a kinetic isotope effect. ''Competitive deuterium washout'' experiments were used to measure kinetic isotope effects on V(max)/K(m) of 2.5 for (S)-glutamate and 3.4 for (R)-glutamate. The ratio of the notably different isotope effects was confirmed by ''double competitive deuterium washout'' experiments. Site-directed mutagenesis was used to generate the mutant C73A and C184A enzymes. In each case the mutant enzymes were inactive as racemases. The two mutant enzymes are, however, capable of catalyzing the elimination of HCl from opposite enantiomers of threo-3-chloroglutamic acid, a process that presumably requires only one enzymic base. This finding indicates that the active sites of the mutant enzymes are intact and that the two cysteines flank the bound substrate molecule. It appears that cysteine-73 is responsible for the abstraction of the C-2 hydrogen from (R)-glutamate and cysteine-184 abstracts the proton from (S)-glutamate in the racemization reaction of the wild-type enzyme.